PROJECT SUMMARY Hepatocellular carcinoma (HCC) accounts for nearly 29,000 deaths annually in the United States alone. However, the molecular mechanisms that drive HCC development remain elusive and current HCC therapies provide negligible clinical benefit. Factors that epigenetically silence an HCC tumor suppressor gene have the potential to promote tumorigenesis and thus may provide novel drug targets for HCC therapies. T o discover such factors, we performed an innovative genome-wide human RNA interference (RNAi) screen to identify factors that mediate epigenetic silencing of the HCC tumor suppressor gene Hedgehog-Interacting Protein (HHIP). HHIP is a negative regulator of Sonic hedgehog (SHH) signaling and loss of HHIP due to epigenetic silencing aberrantly activates SHH signaling, which has been proposed to promote tumor growth in multiple cancers including HCC. One of the factors identified in our screen is CDC-like kinase 1 (CLK1), a dual specificity protein kinase that phosphorylates serine/arginine-rich proteins involved in pre-mRNA splicing. We found that CLK1 transforms cultured immortalized hepatocytes and promotes HCC tumor growth in mouse subcutaneous xenografts, and these effects are dependent upon CLK1 protein kinase activity. Notably, epigenetic silencing of HHIP and CLK1 overexpression occur frequently in HCC patient samples, supporting the clinical relevance of our results. Based on these collective findings, we hypothesize that CLK1 is a driver of HCC and functions by epigenetically silencing the tumor suppressor HHIP. The results of the experiments proposed in this application will establish the role of CLK1 as a driver of HCC, determine the mechanism by which CLK1 promotes tumor growth, and evaluate CLK1 as a drug target for HCC therapy. In Aim 1, we will establish the role of CLK1 in initiation and progression of hepatic tumorigenesis using a series of complementary mouse models that recapitulate characteristic features of HCC. In Aim 2, we will test our hypothesis that CLK1 promotes hepatic tumor growth through epigenetic silencing of HHIP, resulting in aberrant activation of SHH signaling. We will also investigate other mechanisms by which CLK1 may promote tumor growth. In Aim 3, we will evaluate CLK1 as a novel drug target for the development of HCC therapeutics. In preliminary experiments, we have found that inhibition of CLK1 enhances natural killer (NK) cell-mediated eradication of HCC cells. Therefore, we predict that reactivation of HHIP by pharmacological inhibition of CLK1 will: (1) directly inhibit tumor growth by blocking oncogenic SHH signaling, and (2) augment NK cell-mediated eradication of tumor cells. To test these predictions we will determine whether the highly specific CLK1 small molecule inhibitor KH-CB19 can effectively inhibit growth of hepatic tumors and enhance the ability of NK cells to eradicate tumors using a complementary series of immunocompromised and immunocompetent mouse models of HCC. Collectively, the results of the experiments proposed in this application will elucidate a novel druggable pathway that promotes HCC development and evaluate a new approach for treating HCC.